Reduction Potentials of [FeFe]-Hydrogenase Accessory Iron–Sulfur Clusters Provide Insights into the Energetics of Proton Reduction Catalysis

Abstract

An [FeFe]-hydrogenase from Clostridium pasteurianum, CpI, is a model system for biological H activation. In addition to the catalytic H-cluster, CpI contains four accessory iron-sulfur [FeS] clusters in a branched series that transfer electrons to and from the active site. In this work, potentiometric titrations have been employed in combination with electron paramagnetic resonance (EPR) spectroscopy at defined electrochemical potentials to gain insights into the role of the accessory clusters in catalysis. EPR spectra collected over a range of potentials were deconvoluted into individual components attributable to the accessory [FeS] clusters and the active site H-cluster, and reduction potentials for each cluster were determined. The data suggest a large degree of magnetic coupling between the clusters. The distal [4Fe-4S] cluster is shown to have a lower reduction potential (∼ < -450 mV) than the other clusters, and molecular docking experiments indicate that the physiological electron donor, ferredoxin (Fd), most favorably interacts with this cluster. The low reduction potential of the distal [4Fe-4S] cluster thermodynamically restricts the Fd/Fd ratio at which CpI can operate, consistent with the role of CpI in recycling Fd that accumulates during fermentation. Subsequent electron transfer through the additional accessory [FeS] clusters to the H-cluster is thermodynamically favorable.

Spectroscopic and biochemical insight into an electron-bifurcating [FeFe] hydrogenase

“…This arrangement would not be compatible with the previously proposed Fd-binding site being HydC. Instead, Fd would interact with the His-ligated [4Fe-4S] cluster of HydA, as has been proposed for the CpHydA [56]. With the H-cluster in one direction, the FMN in another direction and the Fd-binding site in a third direction, this arrangement temptingly implicates the trinity of iron-sulfur clusters in HydA as a potential bifurcation site.…”

[FeFe]-Hydrogenases: recent developments and future perspectives

“…58 The electron exchange between the active site cofactor, accessory clusters, and external electron donors is relatively well understood. [78][79][80][81] In comparison, proton transfer to the catalytic cofactor has not been shown experimentally yet. Selective proton transfer may well be the reason for the outstanding catalytic performance of [FeFe]-hydrogenases.…”

New Approaches to an Old Problem: Original Techniques in [FeFe]-hydrogenase Research

“…This has been elucidated in the case of the two closely related M3-type enzymes from CpI and CaI as well as the sub-class M2 enzymes from Megasphaera elsdenii (MeHydA) and DdH. [31][32][33][34] In the latter case, redox titrations combined with FTIR and EPR spectroscopy revealed that the redox equilibrium of the diiron site and its pKa is influenced by the oxidation state of the F-clusters. 33 Parallel studies of CaI and MeHydA have shown that the F-clusters affects the catalytic bias of the enzyme.…”